Aim: To investigate the molecular targets of DCLAK11, a novel compound discovered from a series of substituted pyridin-3-amine derivatives, and to characterize its anti-tumor properties in vitro.

Methods: Kinase inhibition was measured by an ELISA assay. Cell viability was assessed with an SRB or a CCK8 assay. The alterations induced by kinase signaling proteins in cancer cells were detected by Western blot. Apoptosis was determined by an Annexin V-PI assay. The following assays were used to evaluate the impact on angiogenesis: wound-healing, Transwell, tube formation and microvessel outgrowth from rat aortic rings.

Results: DCLAK11 was a multi-targeted kinase inhibitor that primarily inhibited the EGFR, HER2, and VEGFR2 tyrosine kinases with IC50 value of 6.5, 18, and 31 nmol/L, respectively. DCLAK11 potently inhibited the proliferation of EGFR- and HER2-driven cancer cells: its IC50 value was 12 and 22 nmol/L, respectively, in HCC827 and HCC4006 cells with EGFR exon deletions, and 19 and 81 nmol/L, respectively, in NCI-N87 and BT474 cells with HER2 amplification. Consistently, DCLAK11 blocked the EGFR and HER2 signaling in cancer cells with either an EGFR or a HER2 aberration. Furthermore, DCLAK11 effectively induced EGFR/HER2-driven cell apoptosis. Moreover, DCLAK11 exhibited anti-angiogenic activity, as shown by its inhibitory effect on the proliferation, migration and tube formation of human umbilical vascular endothelial cells and the microvessel outgrowth of rat aortic rings.

Conclusions: DCLAK11 is a multi-targeted kinase inhibitor with remarkable potency against tyrosine kinases EGFR, HER2 and VEGFR2, which confirms its potent anti-cancer activity in EGFR- and HER2-addicted cancers and its anti-angiogenic activity.

fig3: HCC827 (A) and HCC4006 (B) cells were treated with increasing doses of DCLAK11 for 48 h and apoptotic rate was detected by flow cytometry with Annexin V-PI staining. Data are shown as mean±SD from three independent experiments. cP<0.01 vs control. Western blots were performed to observe the cleaved caspase-3 (Asp175), caspase-3, cleaved PARP and full-length PARP protein expression in HCC827 (C) and HCC4006 (D) cells respectively. The more cleaved caspase-3 and cleaved PARP expression represents for the higher level of apoptosis. Representative data are shown.

Mentions:
Apoptosis is one of the major cellular events that may account for the cell death that results from EGFR signaling deprivation. We therefore measured the number of apoptotic cells by Annexin V-PI dual staining in HCC827 and HCC4006 cells after DCLAK11 treatment. Indeed, apoptosis occurred in a concentration-dependent manner following 48 h of DCLAK11 treatment in both HCC827 and HCC4006 cells without the concomitant occurrence of necrosis (Figure 3). For example, the apoptotic rate of HCC827 cells (Figure 3A) increased from 13.56% (untreated group) to 62.87% (30 nmol/L DCLAK11 treated group); the apoptotic rate of HCC4006 cells (Figure 3B) increased from 6.36% (untreated group) to 33.53% (100 nmol/L DCLAK11 treated group). The occurrence of apoptosis was further supported by the cleavage of caspase-3 and poly ADP-ribose Polymerase (PARP) (Figure 3C and 3D). After DCLAK11 treatment, the full-length caspase-3 and PARP proteins were cleaved to generate their active forms in a concentration-dependent manner.

fig3: HCC827 (A) and HCC4006 (B) cells were treated with increasing doses of DCLAK11 for 48 h and apoptotic rate was detected by flow cytometry with Annexin V-PI staining. Data are shown as mean±SD from three independent experiments. cP<0.01 vs control. Western blots were performed to observe the cleaved caspase-3 (Asp175), caspase-3, cleaved PARP and full-length PARP protein expression in HCC827 (C) and HCC4006 (D) cells respectively. The more cleaved caspase-3 and cleaved PARP expression represents for the higher level of apoptosis. Representative data are shown.

Mentions:
Apoptosis is one of the major cellular events that may account for the cell death that results from EGFR signaling deprivation. We therefore measured the number of apoptotic cells by Annexin V-PI dual staining in HCC827 and HCC4006 cells after DCLAK11 treatment. Indeed, apoptosis occurred in a concentration-dependent manner following 48 h of DCLAK11 treatment in both HCC827 and HCC4006 cells without the concomitant occurrence of necrosis (Figure 3). For example, the apoptotic rate of HCC827 cells (Figure 3A) increased from 13.56% (untreated group) to 62.87% (30 nmol/L DCLAK11 treated group); the apoptotic rate of HCC4006 cells (Figure 3B) increased from 6.36% (untreated group) to 33.53% (100 nmol/L DCLAK11 treated group). The occurrence of apoptosis was further supported by the cleavage of caspase-3 and poly ADP-ribose Polymerase (PARP) (Figure 3C and 3D). After DCLAK11 treatment, the full-length caspase-3 and PARP proteins were cleaved to generate their active forms in a concentration-dependent manner.

Bottom Line:
Furthermore, DCLAK11 effectively induced EGFR/HER2-driven cell apoptosis.Moreover, DCLAK11 exhibited anti-angiogenic activity, as shown by its inhibitory effect on the proliferation, migration and tube formation of human umbilical vascular endothelial cells and the microvessel outgrowth of rat aortic rings.DCLAK11 is a multi-targeted kinase inhibitor with remarkable potency against tyrosine kinases EGFR, HER2 and VEGFR2, which confirms its potent anti-cancer activity in EGFR- and HER2-addicted cancers and its anti-angiogenic activity.

Aim: To investigate the molecular targets of DCLAK11, a novel compound discovered from a series of substituted pyridin-3-amine derivatives, and to characterize its anti-tumor properties in vitro.

Methods: Kinase inhibition was measured by an ELISA assay. Cell viability was assessed with an SRB or a CCK8 assay. The alterations induced by kinase signaling proteins in cancer cells were detected by Western blot. Apoptosis was determined by an Annexin V-PI assay. The following assays were used to evaluate the impact on angiogenesis: wound-healing, Transwell, tube formation and microvessel outgrowth from rat aortic rings.

Results: DCLAK11 was a multi-targeted kinase inhibitor that primarily inhibited the EGFR, HER2, and VEGFR2 tyrosine kinases with IC50 value of 6.5, 18, and 31 nmol/L, respectively. DCLAK11 potently inhibited the proliferation of EGFR- and HER2-driven cancer cells: its IC50 value was 12 and 22 nmol/L, respectively, in HCC827 and HCC4006 cells with EGFR exon deletions, and 19 and 81 nmol/L, respectively, in NCI-N87 and BT474 cells with HER2 amplification. Consistently, DCLAK11 blocked the EGFR and HER2 signaling in cancer cells with either an EGFR or a HER2 aberration. Furthermore, DCLAK11 effectively induced EGFR/HER2-driven cell apoptosis. Moreover, DCLAK11 exhibited anti-angiogenic activity, as shown by its inhibitory effect on the proliferation, migration and tube formation of human umbilical vascular endothelial cells and the microvessel outgrowth of rat aortic rings.

Conclusions: DCLAK11 is a multi-targeted kinase inhibitor with remarkable potency against tyrosine kinases EGFR, HER2 and VEGFR2, which confirms its potent anti-cancer activity in EGFR- and HER2-addicted cancers and its anti-angiogenic activity.